Project Summary Myelodysplastic syndrome (MDS) is a heterogeneous group of pre-leukemic bone marrow failure disorders with a yearly incidence of approximately 13,000 in the U.S. Approximately one third of MDS patients will go on to develop acute myeloid leukemia (AML) with a dismal survival of 5-10%. Standard of care therapy only extends life by a median of 9 months in those requiring treatment and there is no cure for the disease outside of stem cell transplant. As the population ages, this disease will only become more common, thus there is an urgent need for more effective treatments in MDS. MDS pathogenesis is marked by both clonal myeloid proliferation and bone marrow immune dysfunction. In MDS bone marrow, a suppressive immune microenvironment develops in response to initial inflammation and contributes to the proliferation of developing blasts, which are free to grow unchecked by the immune system. In fact, myeloid-derived suppressor cells (MDSCs), myeloid cells which are suppressive to T cell function, have been implicated in disease pathogenesis and progression. The JAK/STAT3 pathway is critical in myeloid differentiation and is aberrantly activated in both myeloblasts and MDSCs, both of which increase as MDS progresses. In order to better understand JAK/STAT3 signaling in MDS, we will use single cell and functional genomic assays to profile STAT3 in primary patient samples, with particular focus on myeloblasts and MDSCs ? two separate but central cells types in leukemic progression of MDS. Given our experience studying single cell phenotype and intracellular signaling in AML, we will adapt our methods to study MDS, with particular focus on p-STAT3. An advantage of this approach is the ability to measure all cells within a sample simultaneously and create single cell signaling profiles for each sample and in response to inhibition. We propose three specific aims to investigate JAK/STAT3 signaling in MDS. In Aim I, the basal and cytokine-induced responses of STAT3 signaling will be measured in banked MDS samples to understand the role of STAT3 signaling in leukemic progression. Aim II will test functional and signaling responses to various inhibitors of the JAK/STAT3 pathway. Finally, Aim III will determine the impact of standard therapy on JAK/STAT3 signaling and STAT3 targets in resistant MDS. Chromatin immunoprecipitation (ChIP-seq) will be used here in order to profile the gene targets of STAT3 in MDS. These studies will significantly contribute to our basic understanding of MDS and the therapeutic potential of the JAK/STAT3 pathway moving forward in this disease. This proposal is ultimately designed to allow Dr. Ferrell to develop further skills in mass cytometry, phospho- flow, immunology techniques and ChIP and to provide him with the critical mentoring and training necessary to become an independent physician-scientist.